1. Field of the Invention
The present invention provides avian adeno-associated virus (AAAV) and vectors derived therefrom. Thus, the present invention relates to AAAV vectors for and methods of delivering nucleic acids to cells of subjects.
2. Background Art
To date, eight AAV isolates (AAVR1-8) have been, characterized and sequenced (2, 4, 19, 20, 25, 32, 51, 56) with AAV2 being the most extensively studied. AAV virions are approximately 20-25 nm in diameter and are composed of a mixture of assembled proteins (VPs) that encapsidate a linear ˜4.7 kb single stranded DNA of plus or minus polarity (7, 43). The genome of AAVs is flanked by inverted terminal repeats (ITRs), which in the case of AAV2 are 145 nucleotides. The ITR is organized as three interrupted palindromes that can fold in an energetically favored T-shaped hairpin structure, which can exist in two orientations, termed flip and flop (42). The ITRs serve as origin of replication and contain cis acting elements required for rescue, integration, excision from cloning vectors and packaging (41, 42, 49 and 58).
The genetic map of the AAVs has been derived primarily from studies of AAV2 but is conserved in all serotypes (26, 27, 29, 36, 42, 45, 46, 58, 60, and 64). Two major open reading frames (rep and cap ORFs) and three transcriptional active promoters (P5, P19, P40) have been identified in the genome of AAV2. The P5 and P19 promoters encode for the nonstructural replication proteins Rep78 and Rep 68 and Rep 52 and Rep 40, respectively. Due to differential splicing, Rep78 and Rep52 have different C termini from Rep68 and Rep40. Transcription initiation from two promoters results in Rep78 and Rep68 having different N termini from Rep52 and Rep 40. The P40 promoter transcribes two alternatively spliced mRNAs. The major mRNA species encodes for the major capsid protein VP3 from a conventional AUG codon and the minor capsid protein VP2 from an upstream in frame ACG codon. The minor mRNA species encodes the entire cap ORF to produce the minor capsid protein VP1 (47). VP1, VP2 and VP3 are found in a ratio of 1:1:10, respectively, and this stoichiometry is generated by the high abundance of one of the mRNA species and the low translation efficiency from an ACG codon in the case of VP2 (14, 47, 55). Previous studies have indicated that VP2 and VP3 are sufficient for particle formation and accumulation of encapsidated ssDNA progeny, while VP1 is required for assembly of highly infectious particles (63, 64).
All four Rep proteins possess NTP binding activity, DNA helicase activity and nuclear localization sequences, however only Rep78/68 possess DNA binding ability (33, 34, 66). Mutant AAV defective for the synthesis of the small Rep proteins (Rep52/40) are able to replicate DNA but no ssDNA progeny is encapsidated (16). The ability of Rep78/68 to bind and nick DNA in a sequence and strand specific manner inside the ITR is essential in every phase of the AAV life cycle, namely DNA replication, AAV gene expression, rescue from the integrated state and self-excision from cloning vectors (29, 35, 44). Nicking of the DNA within the ITR at the terminal resolution site (trs) requires binding of Rep78/68 proteins to a motif composed of tandem repeats of GAGY.
Among AAV serotypes, AAV1, 4, 7 and 8 are believed to be of simian origin while AAV2, 3 and 5 are from humans. AAV6 was found in a human adenovirus preparation and is very similar to AAV1. AAVs have also been reported in other mammalian species including canines, bovine, ovine and equine (8). An avian AAV was first isolated from the Olson strain of quail bronchitis adenovirus (68). It was later found that 50% of adenoviral field isolates from chickens in US and Ireland contained AAAVs serologically indistinguishable from the initial isolate (24). The AAAV was found to be 20 nm in diameter, serologically distinct from AAV1-4, did not agglutinate erythrocytes from several species tested and required adenovirus or herpes virus for replication (5, 68). In addition, AAAV was found to inhibit replication of several avian adenovirus and herpes virus (5, 52, 53). Physicochemical studies revealed that the capsid of AAAV consists of three VP proteins similar to other AAVs. The buoyant density of AAAV in CsCl gradients (1.39-1.44 g/cm3) is similar to what have been reported for all AAVs (6, 30, 68).
The ability of AAV vectors to infect dividing and non-dividing cells and establish long-term transgene expression and the lack of pathogenicity has made them attractive for use in gene therapy applications. Recent evidence has indicated lack of cross competition in binding experiments suggesting that each AAV serotype may have a distinct mechanism of cell entry. Comparison of the cap ORFs from different serotypes has identified blocks of conserved and divergent sequence, with most of the later residing on the exterior of the virion, thus explaining the altered tissue tropism among serotypes (19-21, 48, 56). Vectors based on new AAV serotypes may have different host range and different immunological properties, thus allowing for most efficient transduction in certain cell types. In addition, characterization of new serotypes will aid in identifying viral elements required for altered tissue tropism.
Serological studies have provided evidence of avian adeno-associated virus infection in humans (69). Six percent of an unselected adult population was found positive for antibody to AAAV by agar gel precipitation (AGP), and 15.6% was positive by virus neutralization (VN). Fourteen percent of poultry workers (industry or research) were positive for AAAV antibody by AGP and 66% were positive by VN. In the same studies, no cross reaction was noted by AGP when antiserum to AAAV was reacted against primate antigens of serotypes 1-4 or when antiserum to AAV serotypes 1-4 were reacted against AAAV antigen. In addition, antiserum prepared against primate AAV1-4 did not neutralize the avian AAV. These results show that AAAV is a distinct serotype and infections are not restricted to avian species but are found in the human adult population.
Based on the genome organization and sequence homology among insect densovirus, rodent parvovirus and human dependovirus, it has been previously proposed these virus may have diverged from a common ancestor and evolved strictly in their hosts (3). However, the high sequence homology between avian autonomous parvovirus and primate AAVs and the epidemiological documentation of AAAV transmission to humans provide evidence for host-independent evolution of at least some parvovirus genera. To better understand the relationship between the avian and the primate AAVs, the complete viral genome of AAAV was cloned and sequenced and used to generate recombinant viral particles.
The present invention provides the first complete genomic AAAV sequence. The genome of AAAV is 4,694 nucleotides in length and has similar organization with that of other AAVs. The entire genome of AAAV displays 56-65% identity at the nucleotide level with the other known AAVs. The AAAV genome has inverted terminal repeats of 142 nucleotides with the first 122 forming the characteristic T-shaped palindromic structure. The putative Rep-binding element (RBE) consists of a tandem (GAGY)4 repeat, and the putative terminal resolution site (trs), CCGGT/CG, contains a single nucleotide substitution relative to the AAV2 trs. Surprisingly and in contrast to AAV5, the AAAV ITR can be used as an origin or replication by either AAV5 or AAV2 Rep proteins for packaging. Thus the AAAV ITR can act as a universal ITR. The Rep ORF of AAAV displays 50-54% identity at the amino acid level with the other AAVs, with most of the diversity clustered at the carboxyl and amino termini. Comparison of the capsid proteins of AAAV and the primate dependoviruses indicate divergent regions are localized to surface exposed loops. Despite these sequence differences, recombinant AAAV particles were produced carrying a lacZ reporter gene by co-transfection in 293T cells and transduction efficiency was examined in both chicken primary cells and several cell lines. This unique tropism allows AAAV to be useful as a vector for the development of transgenic animals and also allows for the vaccination of eggs as well as the preparation of recombinant proteins in avian cultures. The exposed regions of AAAV are also sites for insertions of epitopes for the purpose of changing the tropism of the virus or antigen presentation. The present invention shows that AAAV is the most divergent adeno-associated virus described to date, but maintains all the characteristics unique to the genera of dependovirus.
The present invention provides a vector comprising the AAAV virus or a vector comprising subparts of the virus, as well as AAAV viral particles. While AAAV is similar to primate AAVs, the viruses are found herein to be physically and genetically distinct. These differences endow AAAV with some unique properties and advantages which better suit it as a vector for gene therapy or gene transfer applications. As shown herein, AAAV capsid protein, again surprisingly, is distinct from primate capsid protein and exhibits different tissue tropism, thus making AAAV capsid-containing particles suitable for transducing cell types for which primate AAVs are unsuited or less well-suited. AAAV is serologically distinct and thus, in a gene therapy application, AAAV would allow for transduction of a patient who already possesses neutralizing antibodies to primate isolates either as a result of natural immunological defense or from prior exposure to other vectors. AAAV is also useful for gene transfer to other species for the development of transgenic animals or the production of vaccines and recombinant proteins in eggs. Thus, the present invention, by providing these new recombinant vectors and particles based on AAAV, provides a new and highly useful series of vectors.